Detection of rare sequence variants, methods and compositions therefor

Abstract

The present disclosure encompasses methods of error corrected sequencing (ECS) that enable detection of very rare mutations well below the error rate of convention next generation sequencing (NGS). Further, the methods disclosed herein enable multiplex targeting of genomic DNA.

What is claimed is: 
     
       1. A method of identifying a genetic mutation in a biological sample comprising a nucleic acid obtained from a subject, the method comprising:
 a) forming a hybridization complex by hybridizing a primer pool comprising one or more primer pairs specific complementary to one or more regions of interest of the nucleic acid from the biological sample, producing an extension product by extending from an upstream primer of the primer pairs on the hybridization complex to a downstream primer of the primer pairs on the hybridization complex, and generating a ligation product by ligating the extension product to the downstream primer of the primer pairs, wherein the ligation product comprises the regions of interest flanked by sequences required for amplification; 
 b) producing an attaching product by attaching an adapter comprising a random component and an adapter comprising an index sequence to the ligation product from step a) and generating an amplification product by amplifying the attaching product; 
 c) sequencing the amplification product in step b), wherein redundant reads are generated by the sequencing of the amplification product and wherein the redundant reads are grouped by the random component, thereby identifying a consensus sequence from the nucleic acid; and 
 d) comparing the consensus sequence from the nucleic acid to a wild-type reference sequence from the same species of the subject and identifying the genetic mutation in the biological sample if the consensus sequence differs from the reference sequence. 
 
     
     
       2. The method of  claim 1 , wherein the biological sample comprises about 400 to about 800 ng nucleic acid. 
     
     
       3. The method of  claim 1 , wherein the primer pairs of the primer pool in step a) are specific complementary to more than 500 regions of interest in the nucleic acid and the more than 500 regions of interest in the amplification product are sequenced. 
     
     
       4. The method of  claim 1 , wherein unbound primers of the primer pool in step a) are washed away prior to step (b). 
     
     
       5. The method of  claim 1 , wherein each of the adapter comprising a random component and the adapter comprising an index sequence is a Y-shaped adapter. 
     
     
       6. The method of  claim 1 , wherein the adapter comprising a random component and the adaptor comprising an index sequence are attached to the ligation product from step a) via PCR. 
     
     
       7. The method of  claim 1 , wherein the genetic mutation identified in step d) are clinical silent single-nucleotide variations (SNVs).